Network toxicology, machine learning, single-cell analysis, and molecular docking reveal 6PPD/6PPD-Q-associated atrial fibrillation signatures

INTRODUCTION: Atrial fibrillation (AF) is the most prevalent sustained arrhythmia worldwide, and environmental factors are increasingly recognized as contributors to its complex pathogenesis. The tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidation product 6PPD-quinone (6PPD-Q) are emerging environmental contaminants with reported cardiotoxicity; however, their molecular relationship with AF remains unclear. OBJECTIVES: This study aimed to identify potential molecular targets and pathways that link 6PPD/6PPD-Q exposure to AF using an integrated network toxicology and machine learning strategy. METHODS: Multicohort atrial microarray datasets from patients with AF were integrated to identify AF-related genes using differential expression analysis, weighted gene coexpression network analysis, and GeneCards database screening. Putative targets of 6PPD/6PPD-Q were predicted using multiple in silico databases and intersected with AF-related genes. Network toxicology, enrichment analysis, machine learning with SHAP interpretation, single-cell RNA sequencing, and molecular docking were utilized to identify and characterize hub genes. Key targets were validated using qRT-PCR and western blotting in human cardiac fibroblasts. A mouse exposure model was further used to assess AF inducibility, duration, and cardiac gene expression in vivo. RESULTS: Sixteen putative AF-associated target genes of 6PPD/6PPD-Q were identified. Machine learning analysis highlighted four hub genes as key candidate hubs. Single-cell RNA analysis revealed cell type-specific expression, with enrichment in cardiomyocytes and vascular structural cells. Molecular docking predicted favorable binding interactions between 6PPD/6PPD-Q and the key target proteins. In vitro exposure to 6PPD or 6PPD-Q at the tested concentrations upregulated the mRNA and protein levels of all four candidate genes. In vivo exposure significantly increased pacing-induced AF inducibility, prolonged AF duration, and upregulated the expression of all these genes in cardiac tissues. CONCLUSION: These findings suggest that 6PPD and 6PPD-Q may contribute to AF by targeting specific genes and signaling pathways, with DDR1, BCHE, HTR2B, and KCNJ3 identified as core exposure-related hubs.